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buffer in purifying the histidine tagged refolded protein through affinity column, the method was partially modified. Accordingly, the refolding step was performed after purifying the apo-AOx from the inclusion bodies through Ni2+- affinity column. A significant recovery of the functionally active rAOx from inclusion bodies was achieved and the fluorescence data confirmed the successful incorporation of cofactor FAD with the protein matrix. CD study of the rAOx was carried out in presence of glycerol (a critical component of the refolding buffer), which prevented off-pathway aggregation and acted as an osmolyte thus conferred structural stability to our native refolded protein.
Comparing MALDI-TOF/TOF mass of apo-AOx with SDS-PAGE revealed a loss of
~1.6 kDa (mass of N-terminal and C-terminal 6x Histidine tag combined) possibly due to the poor ionization of trypsinized N- and C- terminal peptide fragments and poor detection at detector surface. The pI (6.5±0.1) of the apo-AOx was found to be more basic compared to the pI of other reported aryl rAOx expressed in E.coli (Ruiz-Dueñas et al., 2005). The reason is attributed to the presence of higher basic amino acid residues that constituted ~17% of the total amino acids present in the AOx protein.
The CD spectra are strongly dependent on pH as observed. The protein might have experienced conformational transition to a disordered state at pH below 6.0, which is evident from the increase in magnitude of the negative ellipticity (θ) at around 200 nm (Corrêa and Ramos, 2009). The pH dependent aggregation was apparent due to partial unfolding of the protein, evident from the light scattering observed at acidic pH. However, the aggregation is compensated to some extent by the presence of glycerol as a chaotropic agent. The primary aim of the present CD study with rAOx was to investigate the secondary structure of the protein which was successfully predicted as the ordered structure by comparing the results with the reports available for other similar protein. The time dependent CD analysis and detail light scattering studies were thus not considered in this investigation. We analyzed the effect of pH on the secondary structure conformation and found that the CD profile with a strong negative bands in the range from 200 to 260 nm and signal intensity at ~ 208 nm greater than at 222 nm which is a typical of α+β protein well studied for other proteins as reported by Yang et al, (1996).
Reports on the crystal structures of AOx enzymes from fungal sources are limited with PDB id 1VAO and 3FIM from P. simplicissimum (Mattevi et al., 1997) and P. eryngii (Varela et
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al., 2000a), respectively are the most prominent submissions. Both these crystal structures revealed significant dissimilarities between their 3D structures as well as in their amino acid sequence content. Multiple sequence alignment of A. terreus rAOx with the above aryl AOxs from these lignin degrading strains revealed significant sequence diversity. The cDNA sequence of the aryl AOx from A.terreus deduced by us consisted of 666 amino acids, whereas, the widely studied aryl AOxs from P. pulmonarius, P. eryngii and P. simplicissimum consisted of 594, 593 and 560 amino acid residues, respectively (Varela et al., 1999; 2000a; Benen et al., 1998). The amino acid sequence identity (using NCBI BLAST) of A. terreus AOx with other aryl AOxs from P. pulmonarius, P. eryngii and P. simplicissimum showed 27 %, 25 % and 37 %, respectively. Even with the prevailing sequence variation, the predicted model of A.terreus rAOx showed significant structural homology with chain B of aryl AOx from P. eryngii (PDB id:
3FIM) (Fernández et al., 2009) and its function was that of an aromatic AOx. The Ramachandran plot predicted our modeled protein to be stereo-chemically significant, thus increasing the authenticity of the ab-initio based 3D model. Further validations of our modeled 3D structure were proven through docking simulation studies with its co-factor FAD, which precisely predicted the conserved N-terminal binding region (Rossmann fold; GXGXXG motif, X=any amino acid residue) in our model. The docking also confirmed the β-α-β fold essential for non- covalent interaction with FAD. Function as predicted by I-TASSER was further validated through docking simulations carried out with our modeled rAOx along with four aromatic alcohols used in our kinetic studies. Based on MolDock scoring function the substrates were evaluated on the basis of its binding energies and was found to be consistent with the kinetics studies. The docking studies clearly demonstrated the close proximity of the active substrate binding site and the co-factor FAD binding site separated by a narrow funnel shaped cavity connecting the both. It also confirmed that the active site lies in close vicinity of the FAD isoalloxazine ring. This kind of topology is conserved across all members of Glucose-Methanol- Choline (GMC) oxidoreductase family of proteins and is well reported (Ferreira et al., 2009;
Hernández-Ortega et al., 2011a). Presence of few conserved aromatic amino acid residues (Phe 98 and Tyr 55) near the FAD isoalloxazine ring and substrate binding site could be involved in π-π stacking interaction with FAD isoalloxazine ring, thus stabilizing the co-factor. Molecular
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modeling and docking results gave a visual insight into better understanding on the function and catalytic mechanism of this novel AOx enzyme.
In-vitro refolded rAOx showed an optimum temperature and pH of 30 °C and pH 6.0, respectively for its activity. A range of alcohol substrates for activity of the rAOx was studied and found that only the aryl alcohol substrates showed detectable activity for the recombinant enzyme. Steady-state kinetic parameters of rAOx were evaluated based on its catalytic activity exhibited for the oxidation of four different aromatic alcohols. Kinetic constants pertaining to Vmax, Km, kcat and kcat /Km for each of the substrates tested revealed benzyl alcohol as a poor substrate while 4-methoxybenzyl alcohol (ρ-anisyl alcohol) being the best aromatic alcohol substrate followed by 3-methoxybenzyl alcohol (m-anisyl alcohol) and 3, 4-dimethoxybenzyl alcohol (veratryl alcohol). Variation in activation energies (Ea) observed in temperature range from 20-25°C (704.34 kJ mol-1) and 25-30°C (41.47 kJ mol-1) associated with conformational change in the catalytic site improving affinity.
Conformational deactivation energy (Ead =119 kJ mol-1) predicts a covalent interaction between substrate and enzyme active site leading to thermodynamically stable but catalytically inactive enzyme. A high decimal reduction value (D value) of thermoinactive rAOx predicted enzyme being unstable at higher temperatures. Enzyme half life (t1/2) decreased significantly as temperature exceeds beyond the optimal temperature of 30°C for rAOx. ΔH and ΔG values were found to be constant predicting no change in enzyme heat capacity thus maintaining a stable structure. However, this is the first investigation on the thermoinactivation of recombinant AOx from filamentous fungi and no data exists pertaining to similar studies on the other reported and well studied AOxs for comparative analysis.
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Conclusion and Scope for Future
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